Stefan Washausen

Development of starburst cholinergic amacrine cells in the retina of Tupaia belangeri

“Starburst” cholinergic amacrines specify the response of direction‐selective ganglion cells to image motion. Here, development of cholinergic amacrines was studied in the tree shrew Tupaia belangeri (Scandentia) by immunohistochemistry with antibodies against choline acetyltransferase (ChAT) and neurofilament proteins. Starburst amacrines expressed ChAT much earlier than previously thought. From embryonic day 34 (E34) onward, orthotopic and displaced subpopulations segregated from a single cluster of immunoreactive precursor cells. Orthotopic starburst amacrines rapidly took up positions in the inner nuclear layer. Displaced starburst amacrines were first arranged in a monocellular row in the inner plexiform layer, and, with a delay of 1 week, they descended to the ganglion cell layer. Conversely, dendritic stratification of displaced amacrines slightly preceded that of orthotopic ones. Starburst amacrines expressed the medium‐molecular‐weight neurofilament protein (NF‐M) from E34 to postnatal day 11 (P11) and coexpressed α‐internexin from E36.5 to P11. Consequently, neurofilaments composed of α‐internexin and NF‐M may stabilize developing dendrites of starburst amacrines. During the first 2 postnatal weeks, subpopulations of anti‐NF‐M‐labeled ganglion cells costratified with the preexisting dendritic strata of starburst amacrines in the ON sublamina, OFF sublamina, or both. Hence, anti‐NF‐M‐labeled ganglion cells may include direction‐selective ones. Thereafter, NF‐M and α‐internexin proteins disappeared from starburst amacrines, and NF‐M immunoreactivity was lost in the dendrites of ganglion cells. Our findings suggest that NF‐M and α‐internexin are important for starburst amacrines and ganglion cells to recognize each other and, thus, contribute to the formation of early developing retinal circuits in the inner plexiform layer. J. Comp. Neurol. 502:584–597, 2007.

Rhombomere-specific patterns of apoptosis in the tree shrew Tupaia belangeri

Whether rhombomere-specific patterns of apoptosis exist in the developing hindbrain of vertebrates is under debate. We have investigated the sequence of apoptotic events in three-dimensionally reconstructed hindbrains of Tupaia belangeri (8- to 19-somite embryos). Apoptotic cells were identified by structural criteria and by applying an in situ tailing technique to visualize DNA fragmentation. Seven rhombomeres originated from three pro-rhombomeres. Among pre-migratory neural crest cells in the dorsal thirds of the neural folds, the earliest apoptotic concentrations appeared in the developing third rhombomere (r3). Dorsal apoptotic maxima then persisted in r3, extended from r3 to r2, and also arose in r5. Transverse apoptotic bands increased the total amount of apoptotic cells in odd-numbered rhombomeres first in r3 and, with a delay, also in r5. This sequence of apoptotic events was paralleled by an approximate rostrocaudal sequence of neural crest cell delamination from the even-numbered rhombomeres. Thus, large-scale apoptosis in r3 and r5 helped to establish crest-free zones that segregated streams of migrating neural crest cells adjacent to r2, r4, and r6. The sequence of apoptotic events observed in the dorsal thirds of rhombomeres matches that reported for the chick embryo. Other shared features are apoptotic peaks in the position of a circumscribed ventricular protrusion of fusing parts of the neural folds in r1 and r2, and Y-shaped apoptotic patterns composed of apoptotic maxima in the dorsal and lateral thirds of r1, r2, and r3. These rhombomere-specific patterns of apoptosis may therefore represent a conserved character, at least in amniotes.

Apoptosis contributes to placode morphogenesis in the posterior placodal area of mice.

In the embryonic head of vertebrates, neurogenic and non-neurogenic ectodermal placodes arise from the panplacodal primordium. Whether and how growth processes of the ectodermal layer, changes in the transcriptional precursor cell profile, or positional changes among precursor cells contribute to interplacodal boundary formation is subject to intense investigation. We demonstrate that large scale apoptosis in the multiplacodal posterior placodal area (PPA) of C57BL/6 mice assists in the segregation of otic and epibranchial placodes. Complex patterns of interplacodal apoptosis precede and parallel the structural individualization of high-grade thickened placodes, with the fundamental separation between otic and epibranchial precursor cells being seemingly prevalent. Interplacodal apoptosis between the emerging epibranchial placodes, which express Neurogenin2 prior to their complete structural individualization, comes out most strongly between the epibranchial placodes 1 and 2. Apoptosis then moves from interplacodal to intraplacodal positions in dorsal and, with a delay, ventral parts of the epibranchial placodes. Intraplacodal apoptosis appears to exert corrective actions among premigratory neuroblasts, and helps to eliminate the epibranchial placodes. The present findings confirm and extend earlier observations in Tupaia belangeri (Washausen et al. in Dev Biol 278:86–102, 2005), regarded as an intermediate between primates and other eutherian orders. Having now available maps of apoptosis in the PPA of embryonic mice, further investigations into the functions of inter- and intraplacodal apoptosis can be carried out in an experimentally and genetically more accessible mammalian model organism.

Diversity in mammalian chiasmatic architecture: Ipsilateral axons are deflected at glial arches in the prechiasmatic optic nerve of the eutherian Tupaia belangeri

Permanent ipsilaterally projecting axons approach the chiasmatic midline in rodents but are confined to lateral parts of the optic chiasm in marsupials. Hence, principally different mechanisms were thought to underlie axon pathway choice in eutherian (placental) and marsupial mammals. First evidence of diversity in eutherian chiasmatic architecture came from studies in the newborn and adult tree shrew Tupaia belangeri (Jeffery et al. [ 1998 ] J. Comp. Neurol. 390:183–193). Here, as in marsupials, ipsilaterally projecting axons do not approach the midline. The present study aims to clarify how the developing tree shrew chiasm is organized, how glial cells are arranged therein, and the extent to which the tree shrew chiasm is similar to that of marsupials or other eutherians. By using routinely stained serial sections as well as immunohistochemistry with antibodies against glial fibrillary acidic protein, vimentin, and medium‐molecular‐weight neurofilament protein, we investigated chiasm formation from embryonic day 18 (E18) to birth (E43). From E22 onward, ipsilaterally projecting axons diverged from contralaterally projecting axons in prechiasmatic parts of the optic nerve. They made sharp turns when arriving at glial arches found at the transition from the optic nerve to the chiasm. Thus, during the ingrowth period of axons, Tupaia belangeri and marsupials have specialized glial arrays in common, which probably help to deflect ipsilaterally projecting axons to lateral parts of the chiasm. Our observations provide new evidence of diversity in eutherian chiasmatic architecture and identify Tupaia belangeri as an appropriate animal model for studies on the mechanisms underlying axon guidance in the developing chiasm of higher primates. J. Comp. Neurol. 508:437–457, 2008.

Pax2/Pax8-defined subdomains and the occurrence of apoptosis in the posterior placodal area of mice

The present work aims to improve our understanding of the causes and functions of apoptosis during the morphogenesis of epibranchial placodes in mice. Schematic maps helped to compare the spatiotemporal sequence of apoptotic events with the protein expression patterns of general (Six1) and specific placodal markers (Pax2, Pax8). Our findings challenge the view that, in mammals, all three epibranchial placodes spring from the original posterior placodal area (PPA) of presomite or early somite embryos. Instead, close-meshed analysis of the Pax2/Pax8 expression patterns demonstrates the stepwise emergence of two subdomains which both belong to the gradually expanding PPA, and which largely give rise to the otic placode and epibranchial placode 1 (anterior subdomain), or to the caudal epibranchial placodes (posterior subdomain). Our observations reinforce previous doubts raised on the PPA progeny of early somite Xenopus embryos (Schlosser and Ahrens, Dev Biol 271:439–466, 2004). They also demonstrate that partly different Pax2/Pax8 codes accompany epibranchial placode development in Xenopus laevis and mice. In mice, interplacodal apoptosis assists in the establishment of the two PPA subdomains and, subsequently, of individualized placodes by predominantly eliminating Six1+ placodal precursor cells. Onset of interplacodal and intraplacodal large-scale apoptosis is almost always preceded and/or paralleled by Pax2/Pax8 expression minima in the very same region. Future work will demand the use of knock-out mice and whole embryo culture to experimentally test, whether the combined action of differentially expressed Pax2 and Pax8 genes exerts antiapoptotic effects in the mammalian PPA.

Possibilities and limitations of three-dimensional reconstruction and simulation techniques to identify patterns, rhythms and functions of apoptosis in the early developing neural tube

The now classical idea that programmed cell death (apoptosis) contributes to a plethora of developmental processes still has lost nothing of its impact. It is, therefore, important to establish effective three-dimensional (3D) reconstruction as well as simulation techniques to decipher the exact patterns and functions of such apoptotic events. The present study focuses on the question whether and how apoptosis promotes neurulation-associated processes in the spinal cord of Tupaia belangeri (Tupaiidae, Scandentia, Mammalia). Our 3D reconstructions demonstrate that at least two craniocaudal waves of apoptosis consecutively pass through the dorsal spinal cord. The first wave appears to be involved in neural fold fusion and/or in selection processes among premigratory neural crest cells. The second one seems to assist in establishing the dorsal signaling center known as the roof plate. In the hindbrain, in contrast, apoptosis among premigratory neural crest cells progresses craniocaudally but discontinuously, in a segment-specific manner. Unlike apoptosis in the spinal cord, these segment-specific apoptotic events, however, precede later ones that seemingly support neural fold fusion and/or postfusion remodeling. Arguing with Whitehead that biological patterns and rhythms differ in that biological rhythms depend “upon the differences involved in each exhibition of the pattern” (Whitehead in An enquiry concerning the principles of natural knowledge. Cambridge University Press, London, 1919, p. 198) we show that 3D reconstruction and simulation techniques can contribute to distinguish between (static) patterns and (dynamic) rhythms of apoptosis. By deciphering novel patterns and rhythms of developmental apoptosis, our reconstructions help to reconcile seemingly inconsistent earlier findings in chick and mouse embryos, and to create rules for computer simulations.

Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals

ABSTRACT Placodes are focal thickenings of the surface ectoderm which, together with neural crest, generate the peripheral nervous system of the vertebrate head. Here we examine how, in embryonic mice, apoptosis contributes to the remodelling of the primordial posterior placodal area (PPA) into physically separated otic and epibranchial placodes. Using pharmacological inhibition of apoptosis-associated caspases, we find evidence that apoptosis eliminates hitherto undiscovered rudiments of the lateral line sensory system which, in fish and aquatic amphibia, serves to detect movements, pressure changes or electric fields in the surrounding water. Our results refute the evolutionary theory, valid for more than a century that the whole lateral line was completely lost in amniotes. Instead, those parts of the PPA which, under experimental conditions, escape apoptosis have retained the developmental potential to produce lateral line placodes and the primordia of neuromasts that represent the major functional units of the mechanosensory lateral line system. Summary: Inhibition of apoptosis in mouse embryos reveals rudiments of the lateral line system, a sensory system common to fish and aquatic amphibia, but hypothesized to be completely lost in amniotes.